In analytical chemistry, liquid and gas chromatography techniques have become important tools in the identification of chemical sample components. The basic principle underlying all chromatographic techniques is the separation of a sample chemical compound into individual components by transporting the compound in a moving fluid through a porous retentive media. The moving fluid is referred to as the mobile phase and the retentive media has been referred to as the stationary phase. One of the differences between liquid and gas chromatography is that the mobile phase is either a liquid or a gas, respectively.
Consider, for example, a gas chromatograph. Typically, a supply of inert carrier gas (mobile phase) is continually passed as a stream through a heated column containing porous retentive media (stationary phase). A sample of the subject compound is injected into the mobile phase stream and passed through the column. A detector, positioned by the outlet end of the column, detects each of the separated components as they exit the column. Separation is due primarily to the differential volatility and retention characteristics of each sample component as the temperature in the column is increased.
Gas chromatography detectors typically include heated zones in their base portion which pre-heat the mobile fluid prior to detection at least 50.degree. C. above column temperature to prevent cold trapping solutes in the transfer lines between the column and the detector. In gas chromatography, dimensions of such heated zones are almost irrelevant as long as the fluid is heated sufficiently to prevent cold trapping.
The analytical choice between liquid and gas chromatography techniques is largely dependent of the molecular weight of the compound being analyzed. Liquid chromatographs are capable of analyzing much heavier compounds than gas chromatographs. However, since gas chromatography detection techniques are more sensitive, they are preferred.
The advent of Supercritical Fluid Chromatography (SFC) provided a potential bridge between gas and liquid chromatography advantages, i.e., high sensitivity and heavier molecular weight samples. In SFC, a fluid heated above the critical point, is used as the mobile phase. Such fluid is passed under pressure through a retentive media. As the pressure of the mobile phase is increased, for example, from about 40 ATM to approximately 400 ATM, the sample being analyzed separates into its various components dependent upon the relative differential solubility of each component with the mobile phase. Since the mobile phase is a gas, gas chromatography detectors can be utilized, significantly enhancing detection sensitivity. SFC can be simplistically viewed as an extension of gas chromatography to higher molecular weight components where separation is now density related rather than temperature related. SFC has been found to be primarily useful in the analysis of moderate molecular weight homologous series (M.W. 100 to 10,000) and some thermally labile molecules such as pesticides and drugs.
Originally, SFC systems used media packed columns similar to those used in gas chromatography. However, due to concerns over large pressure drops, decreased efficiency and poor media stability, the use of capillaries as the stationary phase in place of media packed columns has becomes generally accepted. SFC systems utilizing a capillary stationary phase are generally referred to as capillary SFC systems. Systems of the type will find the present invention of primary usefulness.
Consider generally the capillary SFC system. A mobile phase gas is supplied to a pump which in turn supplies the gas to an injector. The sample to be analyzed is injected into the mobile phase and the combination is supplied to a splitter. Since capillary (Stationary Phase) SFC utilizes a very small volume, typically on the order of 100 nanoliters, a splitter is incorporated to ensure that only the allowed volume of the combination is passed through the capillary for analysis. In order to maintain the temperature of the mobile phase above its critical temperature, the splitter and the capillary are placed within an oven. Since SFC is carried out under predetermined pressure conditions to effect various density changes, either static or programmed dynamic pressure conditions, a restrictor is placed at the capillary outlet and in the splitter's excess fluid output to limit mass flow. Pressure is controlled by the pump. The capillary restrictor output is passed through a detector. The output of the detector, a voltage signal, is applied to an X-Y plotter or some form of chart recorder which graphs detector output vs. time. Since pressure is also changed over time, for example, increasing the pump pressure at a fixed rate, the resulting SFC chromatiogram is also a reflection of detector output vs. pressure.
As will be understood, the splitter design and the restrictors function to create a flow ratio such that the nanoliter capillary volume is maintained. This ratio is also referred to as the split ratio.
Since SFC operates at very high pressures and gas chromatography style detectors typically operate near ambient pressure or below, a pressure reduction device must be incorporated. To prevent loss of solutes in a low pressure transfer line, the pressure reduction device should be placed inside the base of the detector so that a portion of the mobil phase expansion, which occurs as gas exits the device, takes place in the detector. Typically, the restrictor placed at the capillary outlet serves to both limit mass flow and to reduce mobile phase pressure prior to detection. The necessity for pressure reduction is also dependent on the type of detector used. For example, if a spectrofluorimetric detector is used, detection would occur prior to the restrictor.
Unfortunately, the design of the restrictor incorporated in such previous SFC systems and the manner in which such restrictors were heated has had an effect on detection efficiency. Consider first the general design characteristics of a typical restrictor which will be similar to that shown in FIG. 1. A restrictor 10, which is of generally cylindrical shape, is provided with a central bore 12 about a central axis 14. Central bore 12 at its inlet end 16 is of substantially the same diameter D.sub.i as the internal diameter of the capillary to which restrictor 10 would be attached. The outlet end 18 is of a substantially smaller diameter D.sub.o than inlet and 16. Beginning a distance L.sub.t upstream from outlet end 18, the interior surface of bore 12 converges forward towards axis 14 yielding a generally frusto conical shape. As used herein, the length L.sub.t is referred to as the taper length. Prior to the present invention, the importance of the taper length has not been appreciated.
Consider now, some specific design characteristics of known restrictors. One previous restrictor incorporated a thick wall capillary drawn down to a pinhole with a very steep taper on the internal diameter having a relatively short taper length. Appropriate dimensions are on the order of exit diameter of 0.5 to 4 .mu.m, inlet diameter of 25 to 100 .mu.m, outside diameter of 300 to 500 .mu.m and taper length of 1 to 5 mm. Total length may be 10 to 20 cm. Alternatively, present restrictors have been integrally formed in an analytical capillary column, for example a drawn restrictor, having a much longer taper length.
Consider now the heating of the mobile phase flowing through above described restrictors in present SFC systems. Since the restrictor internal diameter (ID) just ahead of the taper remains the same as the restrictor inlet, the pressure in this region is the same as column pressure. When using an unmodified gas chromatography detector such as an FID for SFC detection, fluid density can decrease as much as five time as a worst case as a result of the heating caused by the FID heat zone length. This also increases fluid linear velocity as mush as five times. Such a density drop will likely cause solutes to drop out of solution and stick to the walls of the restrictor. For low volatility components the modest increase in linear velocity is unlikely to sweep such components off the restrictor walls and into the detector. The effect will be most severe for higher molecular weight, low volatility solutes which will yield distorted molecular weight distribution information such as desirable in the analysis of homologous series.
Heating restrictors is necessary because restrictors having the dimensions described above cause substantial drops in mobile phase pressure which results in substantial adiabatic cooling of the fluid. The resulting fluid temperature drop is characteristic of the molecular structure. For molecules like carbon dioxide, final temperature occurring after the fluid exits the restrictor, may reach 0.833 times initial, pre-expansion temperature. Phase transition to solid or liquid particles can occur. Such particles can entrap solute molecules and yield noisy flow and signal bursts in the detector as "snow" particles melt. Consequently, SFC detection efficiency will not reach desired levels until the above problems can be resolved.
In the tapered part of the restrictor during a heating operation, continuously decreasing ID combined with increasing temperature and decreasing pressure causes rapid increases in linear velocity. While this helps sweep solutes dropping out of solution into the detector the transit time of individual molecules across the restrictor can becomes extremely short down into the tens of microseconds, complicating heat transfer into the fluid.
The restrictor heating problem is further complicated in that the best materials of construction for restrictors are both chemically and catalytically inert. Fused silica tubing is probably the best material, however, it is also a relatively good insulator. Additionally, restrictors of the type previously described are most easily fabricated using thick wall tubing further decreasing heat transfer.
Some supercritical fluids of interest in chromatography have low critical temperatures. The required restrictor temperatures to avoid phase transitions can therefore also be quite low. It is sometimes true that this temperature is below oven temperature. Consequently, it may be desirable to cool or maintain mobile temperature in the restrictor.
In some situations such as in the use of an unmodified FID, the restrictor effluent is burned in a hydrogen-oxygen or hydrogen-air flame. Such combustion produces water vapor which depending on detector design can condense to a liquid in the exit chimney. During gas chromatography using an FID, the detector base heated zone is quite hot, up to 400.degree. C. and is at least partially thermally coupled to the exit chimney preventing water condensation. When used with SFC, the same detectors can and are run much cooler so that water condensation is much more likely. Water droplets thus formed can run down into the detection volume causing corrosion or even extinguishing the flame. Corrosion causes noisy detector response and flame extinguishment prevents detection.
Consequently, a need still exists for a capillary SFC system which incorporates a restrictor and which heats the mobile phase passing through such restrictor, yet is capable of maintaining a high detection efficiency.